Methods of optimizing drug therapeutic efficacy for treatment of immune-mediated gastrointestinal disorders

ABSTRACT

The present invention provides a method of optimizing therapeutic efficacy and reducing toxicity associated with 6-mercaptopurine drug treatment of an immune-mediated gastrointestinal disorder such as inflammatory bowel disease. The method of the invention includes the step of determining the level of one or more 6-mercaptopurine metabolites in the patient having an immune-mediated gastrointestinal disorder.

This application claims the benefit of priority of provisionalapplication Ser. No. 60/101,714, filed Sep. 24, 1998, which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to autoimmunity andimmune-mediated gastrointestinal disorders such as inflammatory boweldisease and more specifically to methods for optimizing treatment ofimmune-mediated gastrointestinal disorders.

BACKGROUND INFORMATION

Immune-mediated gastrointestinal disorders encompass a wide range ofdebilitating gastrointestinal diseases of various etiologies. One suchimmune-mediated gastrointestinal disorder, inflammatory bowel disease(IBD), is the collective term used to describe two gastrointestinaldisorders of unknown etiology: Crohn's disease (CD) and ulcerativecolitis (UC). The course and prognosis of IBD, which occurs world-wideand is reported to afflict as many as two million people, varies widely.Onset of IBD is predominantly in young adulthood with diarrhea,abdominal pain, and fever the three most common presenting symptoms. Thediarrhea may range from mild to severe and in ulcerative colitis oftenis accompanied by bleeding. Anemia and weight loss are additional commonsigns of IBD. Ten percent to fifteen percent of all patients with IBDwill require surgery over a ten year period. In addition, patients withIBD are at increased risk for the development of intestinal cancer.Reports of an increasing occurrence of psychological problems, includinganxiety and depression, are perhaps not surprising symptoms of what isoften a debilitating disease that strikes people in the prime of life.

6-Mercaptopurine (6-MP) and azathioprine (AZA), a pro-drug that isnon-enzymatically converted to 6-mercaptopurine (6-MP), are 6-MP drugsthat can be used as an effective treatment for inflammatory boweldiseases such as Crohn's disease and ulcerative colitis (KirschnerGastroenterology 115:8.13-821 (1998)). 6-MP can be enzymaticallyconverted to various 6-MP metabolites, including 6-methyl-mercaptopurine(6-MMP) and 6-thioguanine (6-TG) and their nucleotides. 6-TG nucleotidesare thought to be the active metabolite in mediating many of the effectsof 6-MP drug treatment.

Thiopurine methyltransferase (TPMT) is a cytoplasmic enzyme thatpreferentially catalyzes the S-methylation of 6-MP and 6-TG to formS-methylated metabolites such as 6-MMP and 6-methylthioguanine (6-MTG),respectively. TPMT exhibits genetic polymorphism, with 89% of Caucasiansand African Americans having high activity, 11% intermediate activityand 1 in 300 TPMT deficient. Clinical studies with AZA and 6-MP haveshown an inverse relationship between TPMT activity and 6-TGNaccumulation. Patients who less efficiently methylate these thiopurineshave more extensive conversion to 6-TGN, which can lead to potentiallyfatal hematopoietic toxicity. Therefore, patients who have less activeTPMT can be more susceptible to toxic side effects of 6-MP therapy.

Although drugs such as 6-MP and AZA have been used for treating IBD,non-responsiveness and drug toxicity unfortunately complicate treatmentin some patients. Complications associated with 6-MP drug treatmentinclude allergic reactions, neoplasia, opportunistic infections,hepatitis, bone marrow suppression, and pancreatitis. Therefore, manyphysicians are reluctant to treat patients with AZA because of itspotential side effects, especially infection and neoplasia.

Thus, there exists a need to develop methods to optimize the dose of6-mercaptopurine drugs and assess biotransformation in individualpatients to optimize the therapeutic efficacy of 6-mercaptopurine drugswhile minimizing toxic side effects. The present invention satisfiesthis need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides a method of optimizing therapeuticefficacy of 6-mercaptopurine drug treatment of an immune-mediatedgastrointestinal disorder. The method includes the steps ofadministering a 6-mercaptopurine drug to a subject having animmune-mediated gastrointestinal disorder; and determining a level of6-thioguanine in the subject having the immune-mediated gastrointestinaldisorder, where a level of 6-thioguanine less than a level correspondingto about 230 pmol per 8×10⁸ red blood cells indicates a need to increasethe amount of 6-mercaptopurine drug subsequently administered to thesubject and where a level of 6-thioguanine greater than a levelcorresponding to about 400 pmol per 8×10⁸ red blood cells indicates aneed to decrease the amount of 6-mercaptopurine drug subsequentlyadministered to the subject. The methods are directed to treatingimmune-mediated gastrointestinal disorders, including inflammatory boweldiseases (IBD) such as Crohn's disease and ulcerative colitis,lymphocytic colitis, microscopic colitis, collagenous colitis,autoimmune enteropathy, allergic gastrointestinal disease andeosinophilic gastrointestinal disease. In a method of optimizingtherapeutic efficacy of 6-mercaptopurine treatment of IBD, the subjecthaving IBD can be, for example, a pediatric subject. The level of6-thioguanine can be determined, for example, in red blood cells usinghigh pressure liquid chromatography.

The present invention also provides a method of reducing toxicityassociated with 6-mercaptopurine drug treatment of an immune-mediatedgastrointestinal disorder. The method of reducing toxicity associatedwith an immune-mediated gastrointestinal disorder includes the steps ofadministering a 6-mercaptopurine drug to a subject having theimmune-mediated gastrointestinal disorder; and determining a level of a6-mercaptopurine metabolite in the subject having the immune-mediatedgastrointestinal disorder, where a level of the 6-mercaptopurinemetabolite greater than a predetermined toxic level of the6-mercaptopurine metabolite indicates a need to decrease the amount of6-mercaptopurine drug subsequently administered to the subject, therebyreducing toxicity associated with 6-mercaptopurine drug treatment of theimmune-mediated gastrointestinal disorder. In a method of the invention,the 6-mercaptopurine metabolite can be, for example, 6-thioguanine andthe predetermined toxic level of 6-thioguanine can correspond, forexample, to a level of about 400 pmol per 8×10⁸ red blood cells. Wherethe elevated 6-mercaptopurine metabolite is 6-thioguanine, the toxicityassociated with 6-mercaptopurine treatment can be, for example,hematologic toxicity. The 6-mercaptopurine metabolite also can be ametabolite such as 6-methyl-mercaptopurine and the predetermined toxiclevel of 6-methyl-mercaptopurine can correspond, for example, to a levelof about 7000 pmol per 8×10⁸ red blood cells. Where the elevated6-mercaptopurine metabolite is 6-methyl-mercaptopurine, the toxicityassociated with 6-mercaptopurine treatment can be, for example, hepatictoxicity.

Further provided by the invention is a method of optimizing therapeuticefficacy and reducing toxicity associated with 6-mercaptopurine drugtreatment of an immune-mediated gastrointestinal disorder. The methodincludes the steps of administering a 6-mercaptopurine drug to a subjecthaving an immune-mediated gastrointestinal disorder; determining a levelof 6-thioguanine in the subject having the immune-mediatedgastrointestinal disorder; and determining a level of6-methyl-mercaptopurine in the subject having the immune-mediatedgastrointestinal disorder, where a level of 6-thioguanine less than apredetermined minimal therapeutic level indicates a need to increase theamount of 6-mercaptopurine drug subsequently administered to thesubject, thereby increasing therapeutic efficacy; where a level of6-thioguanine greater than a predetermined toxic level of 6-thioguanineindicates a need to decrease the amount of 6-mercaptopurine drugsubsequently administered to the subject, thereby reducing toxicityassociated with 6-mercaptopurine treatment of the immune-mediatedgastrointestinal disorder; and where a level of 6-methyl-mercaptopurinegreater than a predetermined toxic level of 6-methyl-mercaptopurineindicates a need to decrease the amount of 6-mercaptopurine drugsubsequently administered to the subject, thereby reducing toxicityassociated with 6-mercaptopurine treatment of the immune-mediatedgastrointestinal disorder.

In such a method of optimizing therapeutic efficacy and reducingtoxicity associated with 6-mercaptopurine drug treatment of animmune-mediated gastrointestinal disorder, the predetermined minimaltherapeutic level of 6-thioguanine can be, for example, a levelcorresponding to about 230 pmol per 8×10⁸ red blood cells; thepredetermined toxic level of 6-thioguanine can be, for example, a levelcorresponding to about 400 pmol per 8×10⁸ red blood cells; and thepredetermined toxic level of 6-methyl-mercaptopurine can be, forexample, a level corresponding to about 7000 pmol per 8×10⁸ red bloodcells. The level of 6-thioguanine and 6-methyl-mercaptopurine each canbe conveniently determined, for example, in red blood cells using highpressure liquid chromatography. The invention further provides methodsto optimize the therapeutic efficacy of 6-mercaptopurine drug treatmentof a non-IBD autoimmune disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows mercaptopurine metabolism and the structures of6-mercaptopurine (6-MP) metabolites. The initial metabolism of 6-MP iscatalyzed by thiopurine methyltransferase (TPMT), xanthine oxidase (XO),and hypoxanthine phosphoribosyltransferase (HPRT). Further metabolism ofthe thionucleotide is catalyzed by inosine monophosphate dehydrogenase(IMPD) and guanosine monophosphate synthetase (GMPS). The breakdown ofazathioprine to 6-mercaptopurine is nonenzymatic.

FIG. 2 shows 6-mercaptopurine (6-MP) metabolism and the 6-MP metabolitesthat are measured as 6-MP, 6-thioguanine (6-TG) and6-methyl-mercaptopurine (6-MMP) (indicated as “MP,” “TG” and “MMP”inside the base). 6-TG mono-phosphate is converted to the di- andtri-phosphate by monophosphate kinase (MPK) and diphosphate kinase(DPK), respectively. The ribonucleoside diphosphate is converted todeoxyribonucleoside diphosphate by ribonucleotide reductase (RR).

FIG. 3 shows ranges of 6-thioguanine in IBD patients treated with a 6-MPdrug.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of optimizing therapeuticefficacy of 6-mercaptopurine (6-MP) drug treatment of an immune-mediatedgastrointestinal disorder. The method includes the steps ofadministering a 6-MP drug to a subject having an immune-mediatedgastrointestinal disorder; and determining a level of 6-thioguanine(6-TG) in the subject having the immune-mediated gastrointestinaldisorder, where a level of 6-TG less than a level corresponding to about230 pmol per 8×10⁸ red blood cells indicates a need to increase theamount of 6-MP drug subsequently administered to the subject and where alevel of 6-TG greater than a level corresponding to about 400 pmol per8×10⁸ red blood cells indicates a need to decrease the amount of 6-MPdrug subsequently administered to the subject. The methods are directedto treating immune-mediated gastrointestinal disorders, includinginflammatory bowel diseases (IBD) such as Crohn's disease and ulcerativecolitis, lymphocytic colitis, microscopic colitis, collagenous colitis,autoimmune enteropathy, allergic gastrointestinal disease andeosinophilic gastrointestinal disease. In a method of optimizingtherapeutic efficacy of 6-MP treatment of IBD, the subject having IBDcan be, for example, a pediatric subject. The level of 6-TG can bedetermined, for example, in red blood cells using high pressure liquidchromatography (HPLC).

The invention provides methods of optimizing therapeutic efficacy of6-MP drug treatment of an immune-mediated gastrointestinal disorder. Themethods of the invention are particularly useful for treating animmune-mediated gastrointestinal disorder such as IBD, including Crohn'sdisease and ulcerative colitis and subtypes thereof. The methods of theinvention allow the clinician to provide an individually optimizeddosage of a 6-MP drug so as to achieve a target level of a 6-MPmetabolite in a particular patient having an immune-mediatedgastrointestinal disorder, thereby optimizing the effectiveness of 6-MPdrug therapy in the patient. The methods of the invention for optimizingtherapeutic efficacy of 6-MP drug treatment involve determining thelevel of 6-TG in a patient having an immune-mediated gastrointestinaldisorder. As disclosed herein, the level of 6-TG measured in a patienttreated with a 6-MP drug was an indicator of the effectiveness of drugtreatment. A level of at least 230 pmol 6-TG/8×10⁸ red blood cells (RBC)was found in responders to drug therapy (see Examples I and II). Theseresults indicate that determining the level of 6-TG can be used toassess whether a patient has a level of 6-TG that is sufficient toalleviate symptoms of an immune-mediated gastrointestinal disorder suchas IBD, thus optimizing therapeutic efficacy.

As used herein, the term “16-mercaptopurine drug” or “6-MP drug” refersto any drug that can be metabolized to an active 6-mercaptopurinemetabolite that has therapeutic efficacy such as 6-TG. Exemplary6-mercaptopurine drugs as defined herein include 6-mercaptopurine (6-MP)and azathioprine (AZA). As illustrated in FIG. 1, both of 6-MP and AZAcan be metabolized to 6-mercaptopurine metabolites such as the exemplary6-mercaptopurine metabolites shown in FIG. 1, including 6-thioguanine(6-TG), 6-methyl-mercaptopurine (6-MM) and 6-thiouric acid. (Lennard,Eur. J. Clin. Pharmacol. 43:329-339 (1992)).

Other 6-MP drugs include, for example, 6-methylmercaptopurine ribosideand 6-TG (Loo et al., Clin. Pharmacol. Ther. 9:180-194 (1968); O'Dwyeret al., J. Natl. Cancer Inst. 83:1235-1240 (1991); Erb et al., CancerChemother. Pharmacol. 42:266-272 (1998); Lancaster et al., Br. J.Haematol. 102:439-443 (1998); Ingle et al., Am. J. Clin. Oncol. 20:69-72(1997); Evans and Relling, Leuk. Res. 18:811-814 (1994)). 6-TG is aparticularly useful 6-MP drug in patients having high TPMT activity.Patients exhibiting high TPMT activity are expected to more easilyconvert 6-MP drugs such as 6-MP and AZA to 6-MMP (see FIGS. 1 and 2). Asdisclosed herein, high levels of 6-MMP are associated withhepatotoxicity (see Examples I and II). Therefore, patients with highTPMT activity can be more susceptible to toxic effects of 6-MP drugtherapy. By administering 6-TG, which is an active 6-MP metaboliteassociated with therapeutic efficacy (see Examples I and II), thetoxicity that can be associated with conversion of 6-MP to 6-MMP isbypassed.

It is understood that the 6-MP metabolites can be the metabolites shownin FIG. 1 or analogues thereof. As used herein, the term “6-thioguanine”or “6-TG” refers to 6-thioguanine or analogues thereof, includingmolecules having the same base structure, for example, 6-thioguanineribonucleoside, 6-thioguanine ribonucleotide mono-, di- andtri-phosphate, 6-thioguanine deoxyribonucleoside and 6-thioguaninedeoxyribonucleotide mono-, di, and triphosphate. The term “6-TG” alsoincludes derivatives of 6-thioguanine, including chemical modificationsof 6-TG, so long as the structure of the 6-TG base is preserved.

As used herein, the term “6-methyl-mercaptopurine” or “6-MMP” refers to6-methyl-mercaptopurine or analogues thereof, including analogues havingthe same base structure, for example, 6-methyl-mercaptopurineribonucleoside, 6-methyl-mercaptopurine ribonucleotide mono-, di-, andtri-phosphate, 6-methyl-mercaptopurine deoxyribonucleoside, and6-methyl-mercaptopurine deoxyribonucleotide mono-, di- andtri-phosphate. The term “6-MMP” also includes derivatives of6-methyl-mercaptopurine, including chemical modifications of 6-MMP, solong as the structure of the 6-MMP base is preserved.

The methods of the invention relate to treatment of an immune-mediatedgastrointestinal disorder. As used herein, the term “immune-mediatedgastrointestinal disorder” or “immune-mediated GI disorder” refers to anon-infectious disease of the gastrointestinal tract or bowel that ismediated by the immune system or cells of the immune system.Immune-mediated gastrointestinal disorders include, for example,inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerativecolitis, lymphocytic colitis, microscopic colitis, collagenous colitis,autoimmune enteropathy, allergic gastrointestinal disease andeosinophilic gastrointestinal disease.

The methods of the invention are particularly useful for treating IBD,or subtypes thereof, which has been classified into the broad categoriesof Crohn's disease and ulcerative colitis. As used herein, “a subjecthaving inflammatory bowel disease” is synonymous with the term “asubject diagnosed with having an inflammatory bowel disease,” and meansa patient having Crohn's disease or ulcerative colitis. Crohn's disease(regional enteritis) is a disease of chronic inflammation that caninvolve any part of the gastrointestinal tract. Commonly, the distalportion of the small intestine (ileum) and cecum are affected. In othercases, the disease is confined to the small intestine, colon oranorectal region. Crohn's disease occasionally involves the duodenum andstomach, and more rarely the esophagus and oral cavity.

The variable clinical manifestations of Crohn's disease are, in part, aresult of the varying anatomic localization of the disease. The mostfrequent symptoms of CD are abdominal pain, diarrhea and recurrentfever. CD is commonly associated with intestinal obstruction or fistula,which is an abnormal passage between diseased loops of bowel, forexample. Crohn's disease also includes complications such asinflammation of the eye, joints and skin; liver disease; kidney stonesor amyloidosis In addition, CD is associated with an increased risk ofintestinal cancer.

Several features are characteristic of the pathology of Crohn's disease.The inflammation associated with CD, known as transmural inflammation,involves all layers of the bowel wall. Thickening and edema, forexample, typically also appear throughout the bowel wall, with fibrosisalso present in long-standing disease. The inflammation characteristicof CD also is discontinuous in that segments of inflamed tissue, knownas “skip lesions,” are separated by apparently normal intestine.Furthermore, linear ulcerations, edema, and inflammation of theintervening tissue lead to a “cobblestone” appearance of the intestinalmucosa, which is distinctive of CD.

A hallmark of Crohn's disease is the presence of discrete aggregationsof inflammatory cells, known as granulomas, which are generally found inthe submucosa. Some Crohn's disease cases display the typical discretegranulomas, while others show nonspecific transmural inflammation. As aresult, the presence of discrete granulomas is indicative of CD,although the absence of granulomas also is consistent with the disease.Thus, transmural or discontinuous inflammation, rather than the presenceof granulomas, is a preferred diagnostic indicator of Crohn's disease(Rubin and Farber, Pathology (Second Edition) Philadelphia: J.B.Lippincott Company (1994)).

Ulcerative colitis (UC) is a disease of the large intestinecharacterized by chronic diarrhea with cramping abdominal pain, rectalbleeding, and loose discharges of blood, pus and mucus. Themanifestations of ulcerative colitis vary widely. A pattern ofexacerbations and remissions typifies the clinical course of most UCpatients (70%), although continuous symptoms without remission arepresent in some patients with UC. Local and systemic complications of UCinclude arthritis, eye inflammation such as uveitis, skin ulcers andliver disease. In addition, ulcerative colitis and especiallylong-standing, extensive disease is associated with an increased risk ofcolon carcinoma.

Several pathologic features characterize UC in distinction to otherinflammatory bowel diseases. Ulcerative colitis is a diffuse diseasethat usually extends from the most distal part of the rectum for avariable distance proximally. The term left-sided colitis describes aninflammation that involves the distal portion of the colon, extending asfar as the splenic flexure. Sparing of the rectum or involvement of theright side (proximal portion) of the colon alone is unusual inulcerative colitis. The inflammatory process of ulcerative colitis islimited to the colon and does not involve, for example, the smallintestine, stomach or esophagus. In addition, ulcerative colitis isdistinguished by a superficial inflammation of the mucosa that generallyspares the deeper layers of the bowel wall. Crypt abscesses, in whichdegenerated intestinal crypts are filled with neutrophils, also aretypical of ulcerative colitis (Rubin and Farber, supra, 1994).

In comparison with Crohn's disease, which is a patchy disease withfrequent sparing of the rectum, ulcerative colitis is characterized by acontinuous inflammation of the colon that usually is more severedistally than proximally. The inflammation in ulcerative colitis issuperficial in that it is usually limited to the mucosal layer and ischaracterized by an acute inflammatory infiltrate with neutrophils andcrypt abscesses. In contrast, Crohn's disease affects the entirethickness of the bowel wall with granulomas often, although not always,present. Disease that terminates at the ileocecal valve, or in the colondistal to it, is indicative of ulcerative colitis, while involvement ofthe terminal ileum, a cobblestone-like appearance, discrete ulcers orfistulas suggest Crohn's disease.

In addition to IBD, immune-mediated GI disorders also include othergastrointestinal diseases such as lymphocytic colitis; microscopiccolitis; collagenous colitis; autoimmune enteropathy, includingautoimmune enteritis and autoimmune enterocolitis; allergicgastrointestinal disease; and eosinophilic gastrointestinal disease,including eosinophilic gastroenteritis and eosinophilic enteropathy.

Over the past two decades, the histological evaluation of colorectalbiopsies obtained by colonoscopy has expanded the spectrum of chronicIBD. A new group of immune-mediated bowel disorders has emerged,characterized by chronic watery diarrhea, minimal or absent endoscopicfindings, and inflammatory changes in mucosal biopsies. Lymphocyticcolitis, also commonly referred to as microscopic colitis, is aclinicopathological syndrome characterized primarily by lymphocyticinfiltration of the epithelium. Collagenous colitis is defined by thepresence of a collagenous band below the surface epithelium, accompaniedby an increase in inflammatory cell infiltrate (Lazenby et al. Hum.Pathol. 20:18-28 (1989)). These disorders are often associated wishother autoimmune diseases such as rheumatoid arthritis, perniciousanemia, thyroiditis, uveitis and type I diabetes mellitus Clinicianshave used immunosuppressive drugs, including 6-MP, to treat thesedisorders (Deslandres et al. J. Pediatr. Gastroenterol. Nutr. 25:341-346(1997))

Autoimmune enteropathy, including autoimmune enteritis and autoimmuneenterocolitis, is a syndrome of severe secretory diarrhea and markedenterocolitis, in association with diagnostic circulating antibodies toenterocytes (Seidman et al., J. Pediatr. 117:929-932 (1990)). Thissyndrome, most often seen in infancy, can be seen in association withother autoimmune diseases. Complete villous atrophy is associated with asevere inflammatory reaction on small bowel biopsies. Although somecases remit after an extended period of time, most patients die withoutimmunosuppressive therapy, which can include 6-MP drug therapy.

Eosinophilic gastrointestinal disease, including eosinophilicgastroenteritis and eosinophilic enteropathy, is characterized by adense infiltration of eosinophils in one or more areas of thegastrointestinal tract, variable intestinal symptoms, and usually aperipheral eosinophilia (80% of cases). Food allergic, includingallergic gastrointestinal disease, and eosinophilic disorders of thegastrointestinal tract are commonly treated by dietary elimination ofthe offending nutrients. However, both food induced and eosinophilicenteropathies may, in certain circumstances, require corticosteroid andimmunosuppressive therapy, including 6-MP (Russo et al., Pediatric Dev.Path. 2:65-71 (1999)).

The methods of the invention relate to optimizing therapeutic efficacyof 6-MP drug treatment of an immune-mediated GI disorder, including IBDsuch as Crohn's disease and ulcerative colitis and subtypes thereof. Themethods of the invention are particularly useful for treating patientsdependent on steroid therapy for maintenance of remission of disease inCrohn's disease and ulcerative colitis patients. As used herein, thephrase “optimizing therapeutic efficacy of 6-MP drug treatment” refersto adjusting the therapeutic dosage of a 6-MP drug such as 6-MP orazathioprine so that the concentration of a 6-MP metabolite that iscorrelated with effective treatment is maintained. As set forth above,the methods of the invention allow the clinician to provide anindividually optimized dosage of a 6-MP drug so as to achieve a targetlevel of a 6-MP metabolite in a particular patient, thereby optimizingthe effectiveness of 6-MP drug therapy in the patient. Therapeuticefficacy generally is indicated by alleviation of one or more signs orsymptoms associated with the disease. In the case of immune-mediated GIdisorders, in particular IBD, therapeutic efficacy is indicated byalleviation of one or more signs or symptoms associated with thedisease, including, for example, joint pain, arthritis, arthalgia,anorexia, growth failure, fistula closure, abdominal pain, diarrhea,recurrent fever, anemia, weight loss, rectal bleeding, inflammation ofthe intestine, and loose discharges of blood, pus and mucus. Methods fordetermining therapeutic efficacy, in particular for treating IBD, aredisclosed herein in Examples I and II.

Therapeutic efficacy can be readily determined by one skilled in the artas the alleviation of one or more signs or symptoms of the disease beingtreated. In the case of IBD, patients can be analyzed using a Crohn'sdisease activity index (Best et al., Gastroenterology 70:439-444(1976)). IBD patients can also be analyzed using a Harvey-Bradshaw index(HBI) (Harvey and Bradshaw, Lancet 1:514 (1980)). The Harvey-Bradshawindex provides an analytical method for measuring signs or symptoms ofCrohn's disease, including the signs or symptoms of general well-being,abdominal pain, number of liquid stools per day, abdominal mass, andcomplications such as arthralgia, uveitis, erythema nodosum, aphthousulcers, pyoderma gangrenosum, anal fissure, new fistula and abscess. TheHarvey-Bradshaw index is particularly useful when evaluating pediatricpatients.

Previous studies suggested that measurement of 6-MP metabolite levelscan be used to predict clinical efficacy and tolerance to azathioprineor 6-MP (Cuffari et al., Gut 39:401-406 (1996a)). However, it wasunknown what concentrations of 6-MP metabolites correlated withoptimized therapeutic efficacy or with toxicity (Cuffari et al., supra,1996a). As disclosed herein, levels of 6-MP metabolites such as 6-TG and6-MMP were determined and correlated with therapeutic efficacy andtoxicity associated with 6-MP drug therapy (see Examples I and II).

The invention is directed to methods of optimizing therapeutic efficacyof 6-MP drug treatment of an immune-mediated GI disorder by monitoringpredetermined levels associated with therapeutic efficacy or toxicityand adjusting the 6-MP drug dosage so as to maintain an optimized dosethat is efficacious and has reduced toxicity. The methods involveadministering a 6-MP drug such as 6-MP or azathioprine to a subjecthaving an immune-mediated GI disorder and determining a level of a 6-MPmetabolite in the subject having the immune-mediated GI disorder. Themethods of the invention are advantageous in that the dosage of a 6-MPdrug can be adjusted to maximize the efficacy of treating animmune-mediated GI disorder such as IBD while minimizing toxicityassociated with 6-MP drug treatment.

As used herein, the term “6-mercaptopurine metabolite” refers to aproduct derived from 6-mercaptopurine in a biological system. Exemplary6-mercaptopurine metabolites are shown in FIG. 1 and include6-thioguanine (6-TG), 6-methyl-mercaptopurine (6-MMP) and 6-thiouricacid and analogues thereof. For example, 6-MP metabolites include 6-TGbases such as 6-TG, 6-thioguanosine mono-, di- and tri-phosphate; 6-MMPbases such as 6-methyl-mercaptopurine and 6-methyl-thioinosinemonophosphate; 6-thioxanthosine (6-TX) bases such as 6-thioxanthosinemono-phosphate; 6-thioruric acid (6-TUA); and 6-MP bases such as6-mercaptopurine and 6-thioinosine monophosphate (see FIG. 2). Theimmunosuppressive properties of 6-NP are believed to be mediated via theintracellular transformation of 6-MP to its active metabolites such as6-TG and 6-MMP nucleotides. Furthermore, 6-MP metabolites such as 6-TGand 6-MMP were found to correlate with therapeutic efficacy and toxicityassociated with 6-MP drug treatment of IBD patients (see Examples I andII).

The level of a 6-MP metabolite can be determined by methods well knownin the art including, for example, those described in Lilleyman andLennard, Lancet 343:1188-1190 (1994); Lennard and Singleton, J.Chromatography Biomed. Applicat. 583:83-90 (1992); Lennard andSingleton, J. Chromatography 661:25-33 (1994); and Cuffari et al., Can.J. Physiol. Pharmacol. 74:580-585 (1996b)). 6-MP metabolites such as6-TG and 6-MMP can be measured, for example, by collecting red bloodcells and extracting thiobases, for example, 6-MP, 6-TG, 6-TX and 6-MMP,which are released by acid hydrolysis. 6-IMP is converted to a formextractable by phenyl mercury salts (Dervieux and Boulieu, Clin. Chem.44:2511-2525 (1998); Duchesne et al., Proc. Amer. Soc. Clin. Oncol.13:137 (1994a); Duchesne et al., Can. J. Physiol. Pharmacol. 72:197(1994b)). Such an analysis measures the thiobase and its analogues,including ribonucloside, ribonucleotide, deoxyribonucleoside,deoxyribonucleotide thiobases as well as mono-, di- and tri-phosphateanalogues, which have been converted to thiobases.

Acid hydrolyzed extracts can be analyzed by resolving 6-MP metabolitesand measuring their levels. For example, HPLC such as reverse phase HPLCis a useful method for resolving and measuring the levels of 6-MPmetabolites, including 6-MP, 6-TG and 6-MMP (Lennard and Singleton,supra, 1992). Ultraviolet light (UV) detection can be used to measurethe 6-MP metabolites. A particularly useful method of measuring 6-MPmetabolites is isocratic reverse phase HPLC with UV detection (Cuffariet al., supra, 1996b).

Other methods for measuring 6-MP metabolites can also be used. Forexample, ion-pairing HPLC with dual UV-wavelength detection can be usedto measure 6-MP metabolites (Zimm and Strong, Anal. Biochem. 160:1-6(1987)). Additional methods for measuring 6-MP metabolites include, forexample, capillary electrophoresis with laser-induced fluorescencedetection (Rabel et al., Anal. Biochem. 224:315-322 (1995)); anionexchange chromatography and fluorescent detection (Tidd and Dedhar J.Chromatography 145:237-246 (1978)); lanthanum precipitation, acidhydrolysis, back extraction and fluorometric assay (Fletcher andMaddocks, Brit. J. Clin. Pharmacol. 10:287-292 (1980)); thin layerchromatography (Bennet and Allen, Cancer Res. 31:152-158 (1971));precolumn derivatization with the thiol-reactive fluorophoremonobromobimane, treatment with alkaline phosphatase, HPLC resolutionand quantification by fluorometry (Warren and Slordal, Anal. Biochem.215:278-283 (1993)); and enzymatic hydrolysis followed by HPLCseparation and UV detection (Giverhaug et al., Ther. Drug Monit.19:663-668 (1997)). 6-MP metabolites such as 6-TG can also be measuredin DNA by degrading DNA to deoxyribonucleosides, derivatizing deoxy-6-TGwith a fluorophore and resolving on reverse phase HPLC (Warren et al.,Cancer Res. 55:1670-1674 (1995)).

As used herein, the level of a 6-MP metabolite can include the 6-MPmetabolite itself, or the level of the 6-MP metabolite and analoguesthereof. For example, as described above, acid hydrolysis can be used torelease thionucleotides from a sample, resulting in conversion of mono-,di- and tri-phosphates to thiobases. In such an analysis, the level ofseveral analogues of a thionucleotide are measured (see FIG. 2). Forexample, measuring 6-TG can include 6-thioguanosine 5′-mono-, di-, andtri-phosphate, and 6-thiodeoxyguanosine 5′-di-, and tri-phosphate.Measuring 6-MP can include 6-mercaptopurine and 6-thioinosine5′-monophosphate. Measuring 6-MMP can include 6-methylmercaptopurine and6-methylthioinosine 5′-monophosphate, and can also include6-methylthioinosine di- and tri-phosphate, as well as 6-methylthioguanosine.

A particularly useful determination of the level of a 6-MP metabolite isthe median level of the 6-MP metabolite since the distribution of 6-MPmetabolite values is trimodal. Unless otherwise designated, the levelsreferred to herein are median levels. A 6-MP metabolite level can alsobe a mean level, if so desired. Unless otherwise designated, the levelsof 6-MP metabolites referred to herein are values per 8×10⁸ RBC, whetherreported as mean or median values.

6-MP metabolite levels can be conveniently assayed using red blood cellsbecause such cells are readily available from the patient, lack anucleus and are easy to manipulate. However, it should be understoodthat any measurement that allows determination of 6-MP metabolite levelscan be used. For example, leukocytes can be used to measure 6-MPmetabolite levels, which can be correlated with 6-MP metabolite levelsin erythrocytes (Cuffari et al., supra, 1996b). Regardless of the methodemployed to measure 6-MP metabolites, one skilled in the art can readilymeasure 6-MP metabolite levels in a sample, for example, in leukocytesor DNA obtained from a patient, and correlate the level of 6-MPmetabolites to the values disclosed herein, which were determined usingRBC.

For convenience, levels of 6-MP metabolites disclosed herein are givenin terms of an assay with RBC and, therefore, are given as an amount ofa 6-MP metabolite in a given number of RBCs. 6-MP metabolites assayed inRBCs can also be determined relative to the amount of hemoglobin.However, it should be understood that one skilled in the art can measure6-MP metabolite levels in samples other than RBCs and readily correlatesuch levels with 6-MP metabolite levels in RBCs. For example, oneskilled in the art can readily determine levels of a 6-MP metabolite incells such as leukocytes, or cells from the oral mucosa, and in RBCs bymeasuring the level of 6-MP metabolites in both types of cells anddetermining the correspondence between levels of 6-MP metabolites inRBCs and the levels in the other cells. Once a correspondence between6-MP metabolites in RBCs and in a sample has been determined, oneskilled in the art can use that correspondence to measure levels in theother sample and correlate those levels with the levels in RBCsdisclosed herein.

As disclosed herein, the level of 6-TG in an IBD patient treated with a6-MP drug was found to correlate with therapeutic efficacy (see ExamplesI and II). In particular, a median level of 230 pmol 6-TG/8×10⁸ RBC ormore was found in IBD patients who responded to 6-MP drug therapy. Thus,as disclosed herein, a level of at least about 230 pmol 6-TG per 8×10⁸RBC can be a minimal therapeutic level of 6-TG for efficaciouslytreating a patient. Accordingly, a level of 6-TG below about 230pmol/8×10⁸ RBC indicates a need to increase the amount of a 6-MP drugadministered to the patient. A minimal therapeutic level of 6-TG forefficaciously treating a patient also can be, for example, at leastabout 240 pmol per 8×10⁸ RBC; at least about 250 pmol per 8×10⁸ RBC; atleast about 260 pmol per 8×10⁸ RBC; at least about 280 mol per 8×10⁸ RBCor at least about 300 pmol per 8×10⁸ RBC. It is understood that theminimal therapeutic levels of 6-TG disclosed herein are useful fortreating immune-mediated gastrointestinal disorders, including IBD, aswell as non-IBD autoimmune diseases.

The methods of the invention directed to determining whether a patienthas a minimal therapeutic level of a 6-MP metabolite are useful forindicating to the clinician a need to monitor a patient for therapeuticefficacy and to adjust the 6-MP drug dose, as desired. For example, in apatient having less than a minimal therapeutic level of a 6-MPmetabolite such as 6-TG and who also presents as unresponsive to 6-MPdrug therapy or having poor responsiveness to 6-MP drug therapy asmeasured by minimal or no effect on a sign or symptom of the diseasebeing treated, one skilled in the art can determine that the dosage of a6-MP drug should be increased. However, if it is determined that apatient has less than a predetermined minimal therapeutic level of a6-MP metabolite but is responsive to 6-MP therapy, the current dose of6-MP drug can be maintained. Based on measuring 6-MP metabolite levelsand assessing the responsiveness of the patient to 6-MP therapy, oneskilled in the art can determine whether a 6-MP drug dose should bemaintained, increased, or decreased.

Although 6-MP drugs such as 6-MP and azathioprine can be used foreffective treatment of an immune-mediated GI disorder, including IBDssuch as Crohn's disease or ulcerative colitis, administration of suchdrugs can be associated with toxic side effects. Toxicities associatedwith 6-MP drug administration include pancreatitis, bone marrowdepression, allergic reactions and drug hepatitis as well as neoplasmsand infectious complications (Present et al., Annals Int. Med.111:641-649 (1989); Cuffari et al., supra, 1996a). As disclosed herein,various toxicities associated with 6-MP drug treatment, includinghepatic toxicity, pancreatic toxicity and hematologic toxicity,correlate with the level of 6-MP metabolites in a subject administered a6-MP drug (see Examples I and II).

Thus, the present invention also provides a method of reducing toxicityassociated with 6-MP drug treatment of an immune-mediated GI disorder.The method of the invention for reducing toxicity associated with 6-MPdrug treatment includes the steps of administering a 6-MP drug to asubject having an immune-mediated GI disorder; and determining a levelof a 6-MP metabolite in the subject having the immune-mediated GIdisorder, where a level of the 6-MP metabolite greater than apredetermined toxic level of the 6-MP metabolite indicates a need todecrease the amount of 6-MP drug subsequently administered to thesubject, thereby reducing toxicity associated with 6-MP drug treatmentof the immune-mediated GI disorder. In a method of the invention, the6-MP metabolite can be, for example, 6-TG and the predetermined toxiclevel of 6-TG can correspond, for example, to a level of about 400 pmolper 8×10⁸ red blood cells. Where the elevated 6-MP metabolite is 6-TG,the toxicity associated with 6-MP treatment can be, for example,hematologic toxicity, including leukopenia or bone marrow suppression.The 6-MP metabolite also can be a metabolite such as 6-MMP, and thepredetermined toxic level of 6-MMP can correspond, for example, to alevel of about 7000 pmol per 8×10⁸ red blood cells. Where the elevated6-MP metabolite is 6-MMP, the toxicity associated with 6-MP drugtreatment can be, for example, hepatic toxicity.

As disclosed herein, the level of a 6-MP metabolite can be determined ina subject treated with a 6-MP drug and compared to a predetermined toxiclevel of a 6-MP metabolite such as 6-TG or 6-MMP to adjust future 6-MPdrug administration, thereby reducing toxicity in the subject. Forexample, as disclosed herein, levels of 6-TG above about 400 pmol/8×10⁸RBC indicated that a patient was likely to experience toxicity, inparticular hematologic toxicity such as leukopenia (see Examples I andII). Accordingly, a level of 6-TG above about 400 pmol/8×10⁸ RBC can bea predetermined toxic level of 6-TG, which indicates that the amount of6-MP drug subsequently administered should be decreased.

It is understood that, when a patient is determined to have a level of a6-MP metabolite such as 6-TG or 6-MMP higher than a predetermined toxiclevel, one skilled in the art can make a determination as to whether a6-MD drug dose should be decreased. For example, if the level of a 6-MPmetabolite such as 6-TG or 6-MMP is higher than a predetermined toxiclevel, one skilled in the art can monitor for toxic side effects bymeasuring one or more of the toxicities associated with 6-MP drugtreatment, as disclosed herein. As disclosed herein, a level of 6-TGgreater than about 400 pmol per 8×10⁸ RBCs was associated with increasedrisk of leukopenia or bone marrow suppression. Therefore, one skilled inthe art can measure white blood cells (WBC) in a patient having levelsof 6-TG higher than a predetermined toxic level to determine if thepatient is exhibiting signs of reduced WBC counts. If such a patientexhibits signs of leukopenia or bone marrow suppression, the 6-MP drugdose can be reduced. However, if it is determined that a patient haslevels of a 6-MP metabolite higher than a predetermined toxic level butdoes not exhibit signs of leukopenia or other 6-MP drug toxicities, oneskilled in the art can determine that the current 6-MP drug dose can bemaintained. Based on measuring 6-MP metabolite levels and determiningsigns or symptoms of toxicities associated with 6-MP drug treatment, oneskilled in the art can determine whether a 6-MP drug dose should bemaintained or decreased. As such, a level of a 6-MP metabolite higherthan a predetermined toxic level can indicate a need to measure atoxicity associated with 6-MP drug treatment such as measuring WBCs orany of the other signs or symptoms of toxicities associated with 6-MPdrug treatment to determine if the 6-MP drug dose should be adjusted.

Furthermore, it is understood that, when decreasing the 6-MP drug dose,one skilled in the art will know or can readily determine whether the6-MP drug dose should be decreased to a lower dose or whether 6-MP drugadministration should be stopped for some period of time, or terminated.For example, if the clinician determines that 6-MP drug therapy shouldbe stopped for some period of time due to levels of a 6-MP metaboliteexceeding a level predetermined to be toxic, the levels of 6-MPmetabolites can be monitored after stopping 6-MP drug therapy until thelevel of the toxic 6-MP metabolite returns to a safe, non-toxic level.At that time, the clinician can resume 6-MP drug therapy, if desired.

The methods of the invention for reducing toxicity associated with 6-MPdrug treatment of a disease involve comparing a level of a 6-MPmetabolite to a predetermined toxic level of a 6-MP metabolite. Ingeneral, a “predetermined toxic level” of a 6-MP metabolite means alevel of a 6-MP metabolite that has been correlated with one or moretoxicities associated with 6-MP drug treatment. As disclosed herein, apredetermined toxic level of 6-TG can be about 400 pmol per 8×10⁸ RBC. Apredetermined toxic level of 6-TG also can be about 350 pmol per 8×10⁸RBC; 370 pmol per 8×10⁸ RBC; 390 pmol per 8×10⁸ RBC; 425 pmol per 8×10⁸RBC; or 450 pmol per 8×10⁸ RBC. It is understood that the predeterminedtoxic levels of 6-TG disclosed herein are useful for treatingimmune-mediated GI disorders, including IBD, as well as non-IBDautoimmune diseases.

Another 6-MP metabolite useful for predicting the likelihood of toxicityis 6-methyl-mercaptopurine (6-MMP). As disclosed herein, a level ofgreater than about 7000 pmol 6-MMP/8×10⁸ in patients administered a 6-MPdrug was associated with toxicity, in particular hepatotoxicity (seeExamples I and II). These results indicate that the level of 6-MMP canbe used to predict toxicity in a patient treated with a 6-MP drug. Asdisclosed herein, a predetermined toxic level of 6-MMP can be about 7000pmol per 8×10⁸ RBC. A predetermined toxic level of 6-MMP also can beabout 6000 pmol per 8×10⁸ RBC; 6500 pmol per 8×10⁸ RBC; 7500 pmol per8×10⁸ RBC; or 8000 pmol per 8×10⁸ RBC. It is understood that thepredetermined toxic levels of 6-MMP disclosed herein are useful fortreating immune-mediated GI disorders, including IBD, as well as non-IBDautoimmune diseases. According to a method of the invention, if thelevel of 6-MMP is above a predetermined toxic level, the subsequentadministration of a 6-MP drug can be decreased to minimize toxicity.

Further provided by the invention is a method of optimizing therapeuticefficacy and reducing toxicity associated with 6-MP drug treatment of animmune-mediated GI disorder such as IBD. The method includes the stepsof administering a 6-MP drug to a subject having an immune-mediated GIdisorder; determining a level of 6-TG in the subject having theimmune-mediated GI disorder; and determining a level of 6-MMP in thesubject having the immune-mediated GI disorder, where a level of 6-TGless than a predetermined minimal therapeutic level indicates a need toincrease the amount of 6-MP drug subsequently administered to thesubject, thereby increasing therapeutic efficacy; where a level of 6-TGgreater than a predetermined toxic level of 6-TG indicates a need todecrease the amount of 6-MP drug subsequently administered to thesubject, thereby reducing toxicity associated with 6-MP treatment of theimmune-mediated GI disorder; and where a level of 6-MMP greater than apredetermined toxic level of 6-MMP indicates a need to decrease theamount of 6-MP drug subsequently administered to the subject, therebyreducing toxicity associated with 6-MP drug treatment of theimmune-mediated GI disorder.

In such a method of optimizing therapeutic efficacy and reducingtoxicity associated with 6-MP drug treatment of an immune-mediated GIdisorder such as IBD, the predetermined minimal therapeutic level of6-TG can be, for example, a level corresponding to about 230 pmol per8×10⁸ red blood cells; the predetermined toxic level of 6-TG can be, forexample, a level corresponding to about 400 pmol per 8×10⁸ red bloodcells; and the predetermined toxic level of 6-MMP can be, for example, alevel corresponding to about 7000 pmol per 8×10⁸ red blood cells. In amethod of the invention, the subject having an immune-mediated GIdisorder such as IBD can be, for example, a pediatric subject. The levelof 6-TG and 6-MMP each can be conveniently determined, for example, inred blood cells using HPLC.

The methods of the invention are useful for optimizing the amount of a6-MP drug to be administered to a patient with an immune-mediated GIdisorder such as IBD. By measuring the levels of 6-MP metabolites suchas 6-MMP and 6-TG, one skilled in the art can determine the 6-MP drugdosage that will result in optimized therapeutic efficacy and reducedtoxicity when administered to a patient.

As disclosed herein, gender and age differences were observed inpediatric patients treated with 6-MP drug therapy (see Example III).Very little change in 6-MP metabolite levels of 6-TG and 6-MMP was seenfor girls who had gone through puberty (older than age 12). However,boys who had gone through puberty (older than age 14) had a markeddecrease in the level of 6-MMP, suggesting that hormonal changesoccurring during puberty can affect the metabolism of a 6-MP drug.Therefore, the methods of the invention can additionally be used tomonitor 6-MP metabolite levels in adolescents, particularly those goingthrough puberty, in order to optimize therapeutic efficacy or minimizetoxic side effects associated with 6-MP therapy.

As disclosed herein, TPMT genotyping is useful for predicting theeffectiveness of 6-MP therapy in an IBD patient (see Example IV).Heterozygote patients are expected to have lower TPMT activity andshould therefore be monitored for high levels of 6-TG for possible toxiclevels associated with leukopenia or bone marrow suppression. 6-MP drugdoses can be reduced accordingly. Wild type homozygous patients areexpected to have higher TPMT activity and should therefore be monitoredto maintain an effective therapeutic level of 6-TG and to determine ifpatients develop toxic levels of 6-MMP. Homozygous patients deficient inTPMT activity can be treated with lower doses of a 6-MP drug providedthat patients are closely monitored for toxicity such as leukopenia.Therefore, TPMT genotyping can be used to predict patient responsivenessto and potential toxicities associated with 6-MP drug therapy.Furthermore, TPMT genotyping can be combined with other methods of theinvention to both determine TPMT genotype and to monitor 6-MPmetabolites. TPMT genotyping can be particularly valuable whendetermining a starting dose of 6-MP drug therapy but can also be usefulwhen adjusting 6-MP drug doses after therapy has begun.

The invention additionally provides a method of optimizing therapeuticefficacy of 6-MP drug treatment of a non-IBD autoimmune disease. Themethod includes the steps of administering a 6-MP drug to a subjecthaving a non-IBD autoimmune disease; and determining a level of6-thioguanine (6-TG) in the subject having the non-IBD autoimmunedisease, where a level of 6-TG less than a minimal therapeutic levelindicates a need to increase the amount of 6-MP drug subsequentlyadministered to the subject and where a level of 6-TG greater than apredetermined toxic level indicates a need to decrease the amount of6-MP drug subsequently administered to the subject. The level of 6-MMPcan also be monitored in a patient having a non-IBD autoimmune diseaseto determine if the level is higher than a predetermined toxic level of6-MMP.

The methods of the invention can be used to optimize therapeuticefficacy of 6-MP drug treatment of a non-IBD autoimmune disease. Such anon-IBD autoimmune disease can be any non-IBD autoimmune diseasetreatable by a 6-MP drug such as 6-MP or azathioprine and, inparticular, can be a disease such as rheumatoid arthritis, systemiclupus erythematosus, autoimmune hepatitis (chronic active hepatitis) orpemphigus vulgaris.

As used herein, the term “non-IBD autoimmune disease” means a diseaseresulting from an immune response against a self tissue or tissuecomponent, including both self antibody responses and cell-mediatedresponses. The term non-IBD autoimmune disease encompassesorgan-specific non-IBD autoimmune diseases, in which an autoimmuneresponse is directed against a single tissue, including myastheniagravis, vitiligo, Graves' disease, Hashimoto's disease, Addison'sdisease, autoimmune gastritis, and Type I diabetes mellitus. The termnon-IBD autoimmune disease also encompasses non-organ specificautoimmune diseases, in which an autoimmune response is directed againsta component present in several or many organs throughout the body.Non-organ specific autoimmune diseases include, for example, systemiclupus erythematosus, progressive systemic sclerosis and variants,polymyositis and dermatomyositis, and rheumatoid disease. Additionalnon-IBD autoimmune diseases include pernicious anemia, primary biliarycirrhosis, autoimmune thrombocytopenia, and Sjögren's syndrome. Oneskilled in the art understands that the methods of the invention can beapplied to these or other non-IBD autoimmune diseases treatable by a6-MP drug such as 6-MP or azathioprine, or other 6-MP drugs, as desired.Specifically excluded from the term “non-IBD autoimmune disease” arediseases resulting from a graft versus host response and inflammatorybowel diseases such as Crohn's disease or ulcerative colitis.

The methods of the invention are also useful for treating anon-immune-mediated GI disorder autoimmune disease. As used herein, theterm “non-immune-mediated GI disorder autoimmune disease” is a non-IBDautoimmune disease and specifically excludes immune-mediated GIdisorders.

The methods of the invention can be particularly useful for optimizingtherapeutic efficacy of 6-MP drug treatment of rheumatoid arthritis.Rheumatoid arthritis is a chronic systemic disease primarily of thejoints, usually polyarticular, marked by inflammatory changes in thesynovial membranes and articular structures and by muscle atrophy andrarefaction of the bones.

The methods of the invention also can be particularly valuable foroptimizing therapeutic efficacy of 6-MP drug treatment of lupuserythematosus and, in particular, systemic lupus erythematosus (SLE).Systemic lupus erythematosus is a chronic, remitting, relapsinginflammatory, and sometimes febrile multisystemic disorder of connectivetissue. SLE can be acute or insidious at onset and is characterizedprincipally by involvement of the skin, joints, kidneys and serosalmembranes.

Autoimmune hepatitis, also called chronic active hepatitis, also can betreated with a 6-MP drug and the dose optimized using the methods of theinvention. Autoimmune hepatitis is a chronic inflammation of the liveroccurring as a sequel to hepatitis B or non-A, non-B hepatitis and ischaracterized by infiltration of portal areas by plasma cells andmacrophages, piecemeal necrosis, and fibrosis.

The methods of the invention also can be useful for treating pemphigusvulgaris, the most common and severe form of pemphigus, which is achronic, relapsing and sometimes fatal skin disease characterizedclinically by the development of successive crops of vesicles and bullaeand treated by azathioprine. This disorder is characterizedhistologically by acantholysis, and immunologically by serumautoantibodies against antigens in the intracellular zones of theepidermis.

The methods of the invention can also be used to optimize thetherapeutic efficacy of 6-MP drug treatment of graft versus hostdisease, which can occur in transplant patients. Graft versus hostdisease occurs when a transplant patient has an immune reaction to thenon-self transplant organ or tissue. The methods of the invention foroptimizing the therapeutic efficacy of 6-MP drug treatment isparticularly useful for treating heart, kidney and liver transplantrecipients. The methods of the invention can be used to optimizetherapeutic efficacy and/or minimize toxicity associated with 6-MP drugtreatment of a transplant patient.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I 6-Mercaptopurine Metabolite Levels Predict Clinical Efficacyand Drug Toxicity in Pediatric IBD

This example describes measuring 6-MP metabolite levels and correlationwith the response of IBD patients treated with a 6-MP drug.

The levels of the 6-MP metabolites 6-TG and 6-MMP were measured in IBDpatients to whom 6-MP pro-drug was administered, and the relationship of6-MP metabolites to clinical disease activity and drug toxicity wasdetermined. Briefly, blood was sampled (n=89) prior to dailyadministration of 6-MP in 55 IBD patients (CD n=51, UC n=4) receiving1-1.5 mg/kg/day over at least a 4 month period (≧4 mo.). When AZA wasadministered, a conversion factor of 2.07 was used to convert to theequivalent 6-MP dose. Twice as much AZA is administered relative to 6-MPto have an equivalent dose of 6-MP.

Erythrocyte 6-TG, 6-MMP and 6-MP thiobases were measured (pmol/8×10⁸RBC) using reverse phase HPLC. Briefly, blood samples were collected inEDTA (ethylene diamine tetraacetic acid) as anticoagulant. Cells werecentrifuged and washed three times with an equal volume of 0.9% saline.Washed packed cells were stored at −70° C. until analysis was performed.

For acid hydrolysis, 500 μl deionized H₂O, 50 μl thawed red blood cells,40 μl of the appropriate standard or control, 500 μl 3.0 N H₂SO₄, and300 μl 10 mM dithiothreitol (DTT) was added to an 8 ml glass screw captube. The capped tubes were placed in a heating block preheated to 100°C. and hydrolyzed. For 6-MMP, hydrolysis was carried out for 5 hours.For 6-MP and 6-TG, hydrolysis was carried out for 1 hour. After theincubation, tubes were cooled in a room temperature water bath. To tubeshydrolyzed for 5 hours (6-MMP), 400 μl 3.4 N NaOH and 1.0 ml 2 M Trisbuffer, pH 9.0 was added. To tubes hydrolyzed for 1 hour (6-MP/6-TG),450 μl 3.4 N NaOH and 500 μl 2M Tris base was added. A volume of 4 ml0.03% phenylmercuric chloride in methylene chloride was added to eachtube. The tubes were capped and lightly agitated on a bi-directionalrotator (15 min for 6-MMP and 30 min for 6-MP/6-TG) The contents weretransferred to a 15 ml polypropylene centrifuge tube and centrifuged at3500 rpm for 3 min at 10° C. The aqueous phase (top layer) wasdiscarded, and 3.0 ml of the organic phase (bottom layer) wastransferred to a clean 15 ml polypropylene centrifuge tube. The analytesin the organic phase were back extracted by adding 225 μl 0.1 N HCl andlightly mixing on an orbital rotator for 5 min. Following vortexing for30 seconds, the tubes were centrifuged at 3500 rpm for 3 min at 10° C.

For 6-MMP analysis, 50 μl analyte was analyzed on a C18 reverse phasecolumn with the mobile phase containing 1 mM DTT, 2.078% triethylamineand 4% methanol, adjusted to pH 3.2 with concentrated H₃PO₄. For 6-MPand 6-TG analysis, 100 μl analyte was analyzed on a C18 reverse phasecolumn using 0.1 M H₃PO₄ and 1 mM DTT in H₂O as the mobile phase.

Hepatic, pancreatic and hematological tests were obtained every 3months. Clinical remission was defined as a Harvey Bradshaw Index<5 inthose patients off corticosteroids or weaned to a level ofprednisone≦0.4 mg/kg/od (administered every other day). Treatmentfailures were defined as non-responders (HBI>5 or steroid dependence) orcessation of 6-MP due to side effects.

As shown in Table 1, a 6-TG level of >225 pmol per 8×10⁸ RBC wasassociated with remission. The median values shown in the tablesrepresent pmol of the indicated 6-MP metabolite per 8×10⁸ RBC. Excessive6-TG and 6-MMP levels were associated with leukopenia andhepatotoxicity, respectively. Negligible metabolite levels detectednon-compliance as a cause of treatment failure in 2/31 cases. Theseresults indicate that 6-MP metabolite levels predict both clinicalresponsiveness and drug-related toxicity. TABLE 1 Group n median 6-TG6-TG >225 median 6-MMP Responders 58 295 45/58 (78%) 3094 Non- 31  184* 8/31 (26%) 2048 responders Hepatic 7 258 5/7  9211** toxicityPancreatic 6 211 2/6 2342 toxicity Hematologic 6  414+ 5/5 7042 toxicityp Values*<0.001+<0.03‡0 < 0.001**<0.001

These results demonstrate that determining levels of 6-MP metabolites isuseful for predicting efficacy and toxicity of 6-MP drug therapyadministered to IBD patients.

EXAMPLE II 6-Mercaptopurine Metabolite Levels Correlate with Optimal6-MP Therapy in IBD Patients

This example describes prospective examination of the correlation of6-MP metabolite levels with therapeutic response to 6-MP drug therapyand 6-MP drug related toxicity in IBD patients treated with 6-MP.

To obtain additional statistical data on IBD patients treated with a6-MP drug, additional patients and samples were analyzed and combinedwith the data obtained in Example I. Blood was sampled at least once in93 IBD patients followed at Sainte-Justine Hospital IBD Center,Montreal, Canada, who were administered 6-MP drug therapy for at least 4months. The 93 patients were pediatric patients, with 80 diagnosed ashaving CD, 8 diagnosed as having UC, and 5 diagnosed as havingindeterminate colitis (CD or UC). All but 7 patients were given AZA. Thedosages were converted to 6-MP equivalents using a factor of 2.07 asdescribed in Example I. For some patients, two or more samples wereobtained and analyzed. Response to 6-MP was defined by clinicalremission (HBI<5, closed fistula) without corticosteroids. Diseaseactivity and physical exam were ascertained at each clinic visit atwhich 6-MP metabolite levels were determined (clinical evaluationpoint). Hematological, pancreatic and hepatic laboratory parameters wereevaluated simultaneously. Erythrocyte 6-TG and 6-MMP concentrations(pmol/8×10⁸ RBC) were measured by HPLC (Cuffari et al., supra, 1996a).

The results of the analysis of 6-MP metabolites in IBD patients areshown in Table 2. The number of samples corresponds to the number ofdifferent samples obtained from the 93 patients. 6-TG quartile analysis,in which values are determined at 25, 50 and 75% of the data set,revealed that the frequency of response significantly increased atlevels>230 pmol/8×10⁸ RBC (p<0.01). Among patients in relapse, only 28%of patients had 6-TG levels>230 pmol/8×10⁸ RBC. In contrast, 65% ofpatients in remission had 6-TG levels>230 pmol/8×10⁸ RBC (p<0.01).Therefore, erythrocyte 6-TG concentrations were significantly andindependently associated with therapeutic response to 6-MP drug therapy.

The induction and maintenance of remission was found to be optimal at6-TG levels>230 pmol/8×10⁸ RBC. 78% of patients above a median 6-TG of230 pmol/8×10⁸ RBC were responders (see FIG. 3). These results indicatethat a 6-TG value of 230 pmol/8×10⁸ RBC can be used to predict efficacyof drug treatment with a 6-MP drug such as 6-MP or azathioprine. TABLE 2Clinical evaluation Median dose point (n) Median 6-TG Median 6-MMP(mg/kg/day) Remission 106 309* 2600 1.3 Relapse 72 197  1602 1.25 *pvalue    <0.0001 0.3 0.4

Toxicity in the IBD patients treated with a 6-MP drug was alsoevaluated. Toxicity was measured essentially as described previously(Cuffari et al., supra; 1996a). Thirty six patients (39%) experienced anadverse event. Hepatotoxicity was observed in 17% of patients, measuredas the serum level of alanine aminotransferase (ALT) or aspartateaminotransferase (AST) (ALT, AST; exceeding or greater than 2×upperlimit of normal). Leukopenia was observed in 14% of patients (whiteblood cell (WBC)<4000). Pancreatic toxicity was observed in 7% ofpatients (lipase/amylase>2×N). High 6-MMP levels correlatedsignificantly with hepatotoxicity (5463 with hepatotoxicity versus 2177without hepatotoxicity; p=0.04). Leukopenia was observed in only 8%(8/106) of samples from patients in remission, with significantly higher6-TG levels observed in these patients (mean value of 490 in patientswith leukopenia versus mean value of 323 without leukopenia; p<0.04;median values were 342 versus 307, respectively). Therefore, leukopeniadid not correlate with therapeutic efficacy. Furthermore, drug dose (perkg) did not correlate with therapeutic efficacy (see Table 2). However,those patients who do develop leukopenia have a higher 6-TG level thanthe rest of the responder group. These results indicate that 6-TG levelsshould be monitored to avoid potential clinical bone marrow suppressionin responder patients who have high levels of 6-TG.

These results demonstrate a significant correlation between erythrocyte6-TG levels and the therapeutic response to 6-MP drug treatment in IBDpatients. The induction and maintenance of remission was found to beoptimal at 6-TG levels>230 pmol/8×10⁸ RBC. Therefore, monitoring 6-MPmetabolite levels, in particular 6-TG, is useful for determining that atherapeutically effective concentration of 6-MP metabolites ismaintained while treating IBD patients with a 6-MP drug. Monitoring 6-MPmetabolite levels, in particular 6-TG and 6-MMP, is also useful forminimizing 6-MP drug related toxicity.

EXAMPLE III Gender and Age Differences in Metabolism of a 6-MP Drug

This example describes gender, and age differences observed in pediatricpatients treated with 6-MP drug therapy.

Pediatric IBD patients undergoing 6-MP drug therapy were assessed forlevels of 6-MP metabolites. These patients were wild type for TPMT.Patients were assessed based on gender and age as it relates to puberty.Puberty is established at 12 years of age in girls and 14 years of agein boys.

As shown in Table 3, the 6-MMP values are much lower in boys afterpuberty (greater than 14 years). Since the total amount ofthiometabolites is lower, this indicates that either lower doses of 6-MPare used or that there is a difference in the bioavailability of 6-MPafter puberty in males. TABLE 3 6-MP Metabolite Levels in Pediatric IBDPatients 6-TG level 6-MMP level (pmol per (pmol per Number 8 × 10⁸ 8 ×10⁸ Ratio Observed RBC) RBC) 6-MMP/6-TG Girls 39 182.2 3447.0 18.52(0-12 y) Girls 116 217.2 3304.5 12.55 (>12 y) Boys 51 235.0 3681.6 17.24(0-14 y) Boys 104 222.5 1662.3* 6.78* (>14 y)*p < 0.001

These results demonstrate that gender and age can affect the metabolismof 6-MP in pediatric IBD patients undergoing 6-MP drug therapy.

EXAMPLE IV Thiopurine Methyltransferase (TPMT) Genotyping andResponsiveness to 6-MP Drug Therapy

This example describes TPMT genotyping of IBD patients treated with 6-MPdrug therapy.

The genotype of TPMT was determined in IBD patients that were respondersand non responders. Genotyping of TPMT was measured essentially asdescribed previously (Baccichet et al., Leuk. Res. 21:817-823 (1997);Zietkiewicz et al., Gene 205:161-171 (1997)). The data shown in Table 4indicate that patients heterozygous for the TPMT mutation hadsignificantly higher 6-TG levels compared to those patients without themutation. All heterozygote patients were responders to 6-MP. TABLE 4TPMT Genotyping of IBD Patients Heterozygote Normal Responder 100% 55%Non responder  0% 45% mean 6-TG 589* 247 p value*less than 0.0001

TPMT genotyping revealed that 8 of 93 (9%) of patients wereheterozygotes. No homozygous TPMT deficient patients were detected. All8 heterozygotes responded to 6-MP and had 6-TG levels>230 pmol/8×10⁸RBC.

These results demonstrate that TPMT genotyping is useful for predictingthe effectiveness of 6-MP therapy in an IBD patient. Heterozygotepatients are expected to have lower TPMT activity and should thereforebe monitored for high levels of 6-TG for possible toxic levelsassociated with leukopenia or bone marrow suppression. 6-MP drug dosescan be reduced accordingly. Wild type homozygous patients are expectedto have higher TPMT activity and should therefore be monitored tomaintain an effective therapeutic level of 6-TG and to determine ifpatients develop toxic levels of 6-MMP. Homozygous patients deficient inTPMT activity can be treated with lower doses of a 6-MP drug providedthat patients are closely monitored for toxicity such as leukopenia.

All journal articles and references provided herein, in parenthesis orotherwise, are incorporated herein by reference.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the claims.

1. A method of optimizing therapeutic efficacy of 6-mercaptopurine drugtreatment of an immune-mediated gastrointestinal disorder, comprising:(a) administering a 6-mercaptopurine drug to a subject having saidimmune-mediated gastrointestinal disorder; and (b) determining a levelof 6-thioguanine in said subject having said immune-mediatedgastrointestinal disorder, wherein a level of 6-thioguanine less than alevel corresponding to about 230 pmol per 8×10⁸ red blood cellsindicates a need to increase the amount of 6-mercaptopurine drugsubsequently administered to said subject and wherein a level of6-thioguanine greater than a level corresponding to about 400 pmol per8×10⁸ red blood cells indicates a need to decrease the amount of6-mercaptopurine drug subsequently administered to said subject.
 2. Themethod of claim 1, wherein said immune-mediated gastrointestinaldisorder is inflammatory bowel disease (IBD).
 3. The method of claim 2,wherein said subject having IBD is a pediatric subject.
 4. The method ofclaim 1, wherein said immune-mediated gastrointestinal disorder isselected from the group consisting of lymphocytic colitis, microscopiccolitis, collagenous colitis, autoimmune enteropathy, allergicgastrointestinal disease and eosinophilic gastrointestinal disease. 5.The method of claim 1, wherein said level of 6-thioguanine is determinedin red blood cells.
 6. The method of claim 5, wherein said level isdetermined using high pressure liquid chromatography.
 7. A method ofreducing toxicity associated with 6-mercaptopurine drug treatment of animmune-mediated gastrointestinal disorder, comprising: (a) administeringa 6-mercaptopurine drug to a subject having said immune-mediatedgastrointestinal disorder; and (b) determining a level of a6-mercaptopurine metabolite in said subject having said immune-mediatedgastrointestinal disorder, wherein a level of said 6-mercaptopurinemetabolite greater than a predetermined toxic level of said6-mercaptopurine metabolite indicates a need to decrease the amount of6-mercaptopurine drug subsequently administered to said subject, therebyreducing toxicity associated with 6-mercaptopurine drug treatment ofsaid immune-mediated gastrointestinal disorder.
 8. The method of claim7, wherein said immune-mediated gastrointestinal disorder is IBD.
 9. Themethod of claim 8, wherein said subject having IBD is a pediatricsubject.
 10. The method of claim 7, wherein said immune-mediatedgastrointestinal disorder is selected from the group consisting oflymphocytic colitis, microscopic colitis, collagenous colitis,autoimmune enteropathy, allergic gastrointestinal disease andeosinophilic gastrointestinal disease.
 11. The method of claim 7,wherein said 6-mercaptopurine metabolite is 6-thioguanine.
 12. Themethod of claim 11, wherein said predetermined toxic level of6-thioguanine corresponds to a level of about 400 pmol per 8×10⁸ redblood cells.
 13. The method of claim 11, wherein said toxicityassociated with 6-mercaptopurine drug treatment is hematologic toxicity.14. The method of claim 7, wherein said 6-mercaptopurine metabolite is6-methyl-mercaptopurine.
 15. The method of claim 14, wherein saidpredetermined toxic level of 6-methyl-mercaptopurine corresponds to alevel of about 7000 pmol per 8×10⁸ red blood cells.
 16. The method ofclaim 14, wherein said toxicity associated with 6-mercaptopurinetreatment is hepatic toxicity.
 17. The method of claim 7, wherein saidlevel of 6-mercaptopurine metabolite is determined in red blood cells.18. The method of claim 17, wherein said level is determined using highpressure liquid chromatography.
 19. A method of optimizing therapeuticefficacy and reducing toxicity associated with 6-mercaptopurine drugtreatment of an immune-mediated gastrointestinal disorder, comprising:(a) administering a 6-mercaptopurine drug to a subject having saidimmune-mediated gastrointestinal disorder; (b) determining a level of6-thioguanine in said subject having said immune-mediatedgastrointestinal disorder; and (c) determining a level of6-methyl-mercaptopurine in said subject having said immune-mediatedgastrointestinal disorder, wherein a level of 6-thioguanine less than apredetermined minimal therapeutic level indicates a need to increase theamount of 6-mercaptopurine drug subsequently administered to saidsubject, thereby increasing therapeutic efficacy, wherein a level of6-thioguanine greater than a predetermined toxic level of 6-thioguanineindicates a need to decrease the amount of 6-mercaptopurine drugsubsequently administered to said subject, thereby reducing toxicityassociated with 6-mercaptopurine drug treatment of said immune-mediatedgastrointestinal disorder, and wherein a level of6-methyl-mercaptopurine greater than a predetermined toxic level of6-methyl-mercaptopurine indicates a need to decrease the amount of6-mercaptopurine drug subsequently administered to said subject, therebyreducing toxicity associated with 6-mercaptopurine drug treatment ofsaid immune-mediated gastrointestinal disorder.
 20. The method of claim19, wherein said immune-mediated gastrointestinal disorder is IBD. 21.The method of claim 20, wherein said subject having IBD is a pediatricsubject.
 22. The method of claim 19, wherein said immune-mediatedgastrointestinal disorder is selected from the group consisting oflymphocytic colitis, microscopic colitis, collagenous colitis,autoimmune enteropathy, allergic gastrointestinal disease andeosinophilic gastrointestinal disease.
 23. The method of claim 19,wherein said predetermined minimal therapeutic level of 6-thioguanine isa level corresponding to about 230 pmol per 8×10⁸ red blood cells. 24.The method of claim 19, wherein said predetermined toxic level of6-thioguanine is a level corresponding to about 400 pmol per 8×10⁸ redblood cells.
 25. The method of claim 19, wherein said predeterminedtoxic level of 6-methyl-mercaptopurine is a level corresponding to about7000 pmol per 8×10⁸ red blood cells.
 26. The method of claim 19, whereinsaid predetermined minimal therapeutic level of 6-thioguanine is a levelcorresponding to about 230 pmol per 8×10⁸ red blood cells, saidpredetermined toxic level of 6-thioguanine is a level corresponding toabout 400 pmol per 8×10⁸ red blood cells, and said predetermined toxiclevel of 6-methyl-mercaptopurine is a level corresponding to about 7000pmol per 8×10⁸ red blood cells.
 27. The method of claim 19, wherein saidlevel of 6-thioguanine and said level of 6-methyl-mercaptopurine each isdetermined in red blood cells.
 28. The method of claim 27, wherein saidlevel is determined using high pressure liquid chromatography.
 29. Themethod of claim 19, wherein said toxicity associated with6-mercaptopurine drug treatment is selected from the group consisting ofhepatic toxicity and hematologic toxicity.
 30. A method of optimizingtherapeutic efficacy of 6-mercaptopurine drug treatment of a non-IBDautoimmune disease, comprising: (a) administering a 6-mercaptopurinedrug to a subject having said non-IBD autoimmune disease; and (b)determining a level of 6-thioguanine in said subject having said non-IBDautoimmune disease, wherein a level of 6-thioguanine less than a minimaltherapeutic level indicates a need to increase the amount of6-mercaptopurine drug subsequently administered to said subject andwherein a level of 6-thioguanine greater than a level corresponding to apredetermined toxic level indicates a need to decrease the amount of6-mercaptopurine drug subsequently administered to said subject.
 31. Themethod of claim 30, wherein said minimal therapeutic level is about 230pmol per 8×10⁸ red blood cells.
 32. The method of claim 30, wherein saidpredetermined toxic level is about 400 pmol per 8×10⁸ red blood cells.33. The method of claim 30, wherein said level of 6-mercaptopurinemetabolite is determined in red blood cells.
 34. The method of claim 33,wherein said level is determined using high pressure liquidchromatography.